title: Increase of the intracellular Ca2+ concentration mediated by transport of glutamate into rat hippocampal synaptosomes: characterization of the activated voltage sensitive Ca2+ channels.
abstract: The changes in the intracellular free Ca2+ concentration, [Ca2+]i, mediated by glutamate and D-aspartate into rat hippocampal synaptosomes was studied. Glutamate increased the [Ca2+]i in a dose-dependent manner with an EC50 of 1.87 microM and a maximal increase of 31.5 +/- 0.9 nM. We also observed that stimulation of the synaptosomes with 100 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), 100 microM kainate, or 100 microM D-aspartate increased the synaptosomal [Ca2+]i. The effect of either of these non-NMDA receptor agonists and of D-aspartate was additive, suggesting the activation of two different components (the ionotropic non-NMDA receptors or the glutamate transporters). Stimulation of synaptosomes with 100 microM glutamate increased the [Ca2+]i and prevented the effect of either non-NMDA receptor agonists and the effect of D-aspartate. We also observed that incubation of the synaptosomes with D-aspartate induced the Ca(2+)-independent release of glutamate, possibly through the reversal of the glutamate carrier. The aim of incubating the synaptosomes with D-aspartate was to avoid undesirable secondary activation of glutamate receptors. After incubating the synaptosomes with 100 microM D-aspartate (10 min at 37 degrees C), the subsequent stimulation with D-aspartate increased the [Ca2+]i due to glutamate transport. This increase in [Ca2+]i induced by 100 microM D-aspartate was insensitive to 1 microM nitrendipine, but was inhibited by about 50% by the presence of both 500 nM omega-CgTx GVIA and 100 nM omega-Aga IVA or by 500 nM omega-CgTx MVIIC. We clearly identified two different processes by which glutamate increased the [Ca2+]i in rat hippocampal synaptosomes: activation of non-NMDA receptors and activation of the glutamate transporters. We also characterized the voltage sensitive Ca2+ channels (VSCC) activated as a consequence of the glutamate transport, and determined that class B (N-type) and class A (P or Q-type) Ca2+ channels were responsible for about 50% of the signal.
authors.affiliation: Center for Neurosciences of Coimbra, Department of Zoology, University of Coimbra, Portugal. jomalva@cygnus.ci.uc.pt
authors.forename: J O
authors.initials: JO
authors.lastname: Malva
authors.forename: A F
authors.initials: AF
authors.lastname: Ambrósio
authors.forename: A P
authors.initials: AP
authors.lastname: Carvalho
authors.forename: C M
authors.initials: CM
authors.lastname: Carvalho
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000502:physiology; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001224:Aspartic Acid / Q000494:pharmacology; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism*; D007608:Kainic Acid / Q000494:pharmacology; D008297:Male; D010455:Peptides / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017470:Receptors, Glutamate / Q000502:physiology; D013111:Spider Venoms / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology; D020867:omega-Agatoxin IVA; D020866:omega-Conotoxin GVIA; D020960:omega-Conotoxins
chemical_list: D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010455:Peptides; D017470:Receptors, Glutamate; D013111:Spider Venoms; D020867:omega-Agatoxin IVA; D020960:omega-Conotoxins; C075851:omega-conotoxin-MVIIC; D001224:Aspartic Acid; D018698:Glutamic Acid; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D020866:omega-Conotoxin GVIA; D007608:Kainic Acid; D002118:Calcium